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1、The S100 calcium-binding protein A11 promotes hepatic steatosisthrough RAGE-mediated AKT-mTOR signalingFei Tenga,b,1,2,Jingjing Jiangc,1,2,Jinhua Zhangd,1,2,Youwen Yuana,b,Kangli Lia,b,Bing Zhouc,Xuan Zhoue,Wenhui Liua,b,Peizhen Zhanga,b,Deying Liua,b,Minghua Zhengf,g,Yan Luc,Huijie Zhanga,b,aDepart
2、ment of Endocrinology and Metabolism,Nanfang Hospital,Southern Medical University,Guangzhou,ChinabGuangdong Provincial Key Laboratory of Shock and Microcirculation,Guangzhou,ChinacThe Key Laboratory of Metabolism and Molecular Medicine,the Ministry of Education,Department of Endocrinology and Metabo
3、lism,Zhongshan Hospital,Fudan University,Shanghai,ChinadKey Laboratory of Functional and Clinical Translational Medicine,Department of General Medicine,Xiamen Medical College,Xiamen,ChinaeThe First Affiliated Hospital of Xiamen University,Xiamen,ChinafMAFLD Research Center,Department of Hepatology,T
4、he First Affiliated Hospital of Wenzhou Medical University,Wenzhou,Zhejiang,ChinagKey Laboratory of Diagnosis and Treatment for The Development of Chronic Liver Disease,Zhejiang Province,Wenzhou,Zhejiang,Chinaa b s t r a c ta r t i c l ei n f oArticle history:Received 22 November 2020Accepted 2 Febr
5、uary 2021Keywords:S100A11Obesity,NAFLDLipid metabolismAKTmTORRationale:Nonalcoholic fatty liver disease(NAFLD),the mostcommoncauseofchronicliverdisease,has becomeanincreasinglyseverepublichealthproblem.However,theunderlyingmechanismfortheoccurrenceanddevel-opment of NAFLD remains largely unknown.S10
6、0 calcium-binding protein A11(S100A11)is a multifunctionalprotein previously reported to be a poor prognostic indicator of hepatocellular carcinoma,while the role ofS100A11 affects NAFLD is still not clear.Methods:Immunohistochemical staining was performed using human NAFLD and control biopsy specim
7、ens.Serum level of S100A11 were analyzed by Elisa assays.The S100A11 over-expressed/knocked-down modelwas established in vitro or in vivo.The expression levels of genes related to lipid metabolism in liver tissuewere performed by quantitative PCR and western blotting.Hepatic lipid accumulation was d
8、etermined by bio-chemical measurements and histochemistry.Results:We showed that the concentration of serum S100A11 was significantly elevated in NAFLD patients,andexpressionofS100A11wasremarkedlyincreasedintheliversofNAFLDpatientsandmousemodels.Overexpres-sionofS100A11invivomarkedlyincreasedliverst
9、eatosis,bodyweight,andserumaspartateaminotransaminase(AST)levels.Mechanistically,our results demonstrated that S100A11 acted as a positive regulator of AKT/mTORsignaling to induce lipid synthesis and aggravate lipid deposition.Conclusions:These results provide evidence for a novel role of S100A11 th
10、at contributes to hepatic steatosis,suggesting that targeting S100A11 may be an alternative approach for the treatment of NAFLD.2021 Elsevier Inc.All rights reserved.1.IntroductionNonalcoholic fatty liver disease(NAFLD)is the leading cause ofchronic liver disease and has become an increasingly sever
11、e publichealth problem1,2.The underlying mechanism for the occurrenceand development of NAFLD is complex and multifactorial.Intracellu-lar accumulation of heterotopic triglycerides(TG)is considered to bean early stage marker of NAFLD,defined as the accretion of TG in theliver exceeding 5%of liver we
12、ight 3.It has been well establishedthat lipogenesis is a prominent anomaly in NAFLD and a key eventleading to massive steatosis 4,although the molecular basisremains to be determined.Various factors such as oxidative stress,mitochondrial dysfunction,and endoplasmic stress play essentialroles in the
13、development of the disease into NAFLD 5.Recent stud-ies have confirmed that cytokines derived from metabolic organs,Metabolism Clinical and Experimental 117(2021)154725Abbreviations:NAFLD,nonalcoholic fatty liver disease;S100A11,s100 calcium-binding protein A11;AST,aspartate aminotransaminase;RAGE,r
14、eceptor for advancedglycation end products;TG,triglycerides;NAFL,nonalcoholic fatty liver;NASH,nonalco-holic steatohepatitis;NAS,NAFLD activity score;Ad,adenovirus;AAV8,adeno-associated virus 8;ALT,alanine aminotransferase;ITT,Insulin tolerance test;GTT,Glucose tolerance test;OCT,optimal cutting tem
15、perature compound;ORO,Oil red O;RT,room temperature;H&E,hematoxylin and eosin;HFD,high-fat diet;NCD,normal chowdiet;PA,palmitic acid;AUC,under the curve;KEGG,Kyoto Encyclopedia of Genes andGenomes.Correspondence to:Y.Lu,Department of Endocrinology and Metabolism,ZhongshanHospital,Fudan University,18
16、0 Fenglin Road,Shanghai 200032,China.Correspondenceto:H.Zhang,DepartmentofEndocrinologyandMetabolism,NanfangHospital,Southern Medical University,1838 North Guangzhou Road,Guangzhou 510515,China.E-mail addresses:lu.yan2zs-(Y.Lu),huijie_(H.Zhang).1These authors equally contributed to this work.2The au
17、thors have no conflicts of interest.https:/doi.org/10.1016/j.metabol.2021.1547250026-0495/2021 Elsevier Inc.All rights reserved.Contents lists available at ScienceDirectMetabolism Clinical and Experimentaljournal homepage:Downloaded for Anonymous User(n/a)at Shanghai Jiao Tong University School of M
18、edicine from ClinicalK by Elsevier on April 04,2021.For personal use only.No other uses without permission.Copyright 2021.Elsevier Inc.All rights reserved.including the liver and adipose tissue,play a crucial role in the devel-opment of NAFLD 6,7.S100 protein,a low molecular weight acidic cytokine,h
19、as been re-ported to play a crucial role in many diseases.It plays important rolesinmultiplebiologicalprocesses,suchasinflammation,autoimmunedis-eases,and tumorigenesis 810.A total of 25 calcium-binding S100family members have been currently identified 11.As reported byLiu et al.,S100A9 can effectiv
20、ely predict pathological liver features,in-cluding steatosis and inflammation 12.Mukai et al.found that serumS100A8 levels were significantly higher in patients with nonalcoholicfatty liver(NAFL)13 and were even higher than in patients with non-alcoholic steatohepatitis(NASH).In addition,S100A4 leve
21、ls in liver tis-sues are positively correlated with liver fibrosis 14.These resultssuggest that the S100 protein family is closely related to the develop-ment of chronic liver diseases.S100A11,another member of the S100 protein family,is alsoknown as S100C.Hitherto,studies on S100A11 have mainly foc
22、usedon its effects on the progression of tumors 1517.Moreover,S100A11 is known as an inducer of inflammatory processes,as evi-denced by its upregulation in various inflammatory diseases,suchas infective endocarditis 18,idiopathic inflammatory myopathies19,and rheumatoid arthritis 20.Furthermore,prev
23、ious studieshave reported that S100A11 levels in liver tissues are positively cor-related with liver fibrosis and hepatocellular carcinoma15,21.However,it remains unclear whether S100A11 is associated withthe development of NAFLD.In this study,we found that S100A11 is upregulated in the livers ofhum
24、an patients and obese mice with NAFLD.We report that S100A11can promote lipid synthesis and aggravate lipid deposition in vivo andin vitro by activating the AKT/mTOR signaling pathway axis throughbinding to the multiligand receptor for RAGE.Our results suggest thatS100A11 can be used not only as a t
25、herapeutic target but also as a po-tential predictive biomarker to monitor the progress of NAFLD inhumans.2.Methods2.1.Human samplesA total of 217 NAFLD patients and 172 healthy controls were in-cluded in our study.The NAFLD group consisted of 110 biopsy-provenNAFL patients and 107 biopsy-proven NAS
26、H patients as described inour previous studies22,23.The diagnosis of NAFLD was based on thefollowingfourhistopathologicalfeatures,includinghepaticmacrovesicular steatosis,hepatocellular ballooning,lobular inflamma-tionandstageoffibrosis.TheNAFLDactivityscore(NAS)wascalculatedaccording to the Kleiner
27、 scoring system.Serum samples were obtainedfrom healthyvolunteers and NAFLDpatients.TheclinicalcharacteristicsofparticipantsarepresentedinTable1.Allsubjectsprovidedwrittenin-formed consent for this study.The human study was approved by theClinical Ethics Committee of First Affiliated Hospital of Wen
28、zhou Medi-cal University(Wenzhou,China),in line with the ethical guidelines ofthe 1975 Declaration of Helsinki.The methods were carried out in ac-cordance with the approved guidelines.2.2.Immunohistochemical assessmentHumanliverbiopsies(0.51cm3)werefixedin10%neutralbufferedformalin,dehydrated,and em
29、bedded in paraffin.The sections weredeparaffinized and rehydrated,and endogenous peroxidase activityand background binding were inhibited using 3%hydrogen peroxidein methanol and 1%bovine serum,respectively.The slides wereimmunoprobed with S100A11 antibodies(Proteintech,Chicago,USA)in a dilution of
30、1:300.Images were taken using an inverted microscopeunder 200 magnification(each group contained three samples).2.3.Measurement of serum S100A11Serum S100A11 levels were measured using a commerciallyavailable ELISA kit according to the manufacturers protocol(Circulex,Nagano,Japan).The detection limi
31、t of the assay was 0.015 ng/ml.2.4.Cell cultureAML12 cells were cultured in DMEM/F12(Gibco)supplementedwith 10%fetal bovine serum(FBS,Biowest),1%antibiotics(Gibco),ITSLiquid Media Supplement(10 g/ml insulin,5.5 g/ml transferrin,and5 ng/ml selenium,Sigma),and 40 ng/ml dexamethasone(Sigma),andincubate
32、d at 37 C in a 5%CO2atmosphere.The cell medium was re-placed every two days.Before transfection,cells were evenly seededonto 6-well plates and incubated for 24 h,then the culture mediumwas discarded,and 100 nmol/l of S100a11-overexpressing adenovirusor empty vector adenovirus(Ad-S100a11;Ad-NC;Genech
33、em,Shang-hai,China)for overexpressing S100a11,and 200 nmol/l of siRNAtargeted to S100a11(si-S100a11),Rage(si-Rage),Cd36(si-Cd36)orcorresponding negative control(si-NC)purchased from Genechem forknocking-down S100a11,Rage,Cd36 expression,were transferredinto cells using Lipofectamine 2000(Invitrogen,
34、Carlsbad,CA,USA)ac-cording to the manufacturers instructions.Rapamycin was purchasedfrom APE*BIO(10 nM for 72 h).The siRNAs sequences targetingS100a11,Rage,Cd36 were as follows:si-S100a11 sense,5-GUGUCCUUGACCGCAUGAUTT-3,and anti-sense,5-AUCAUGCGGUCAAGGACACTT-3;si-Rage sense,5-GGGCAUUC AGCUG UUGGUUTT
35、-3,andanti-sense,5-AACCAACAGCUGAAUGCCCTT-3;si-CD36sense,5-CAGUCGGAGACAUGCUUAUTT-3,and anti-sense,5-AUAAGCAUTable 1Baseline characteristics of the study individuals.VariablesNon-NAFLDNAFLDNASHSample size172110107Age(years)a38.2 10.542.3 11.242.5 11.0Gender(female,%)25(14.5)21(19.1)21(19.6)BMI(kg/m2)2
36、3.3 2.126.0(24.528.0)27.1(24.629.4)Waist circumference(cm)81.9 6.290.8 8.094.9 8.9Systolic BP(mmHg)116.6 10.2127.9 15.1129.8 14.0Diastolic BP(mmHg)72.6 7.183.5 10.084.8 10.4WBC(109/L)6.79 1.516.04 1.746.41 1.47RBC(*1012/L)4.91 0.335.01 0.485.06 0.52HGB(g/L)147.77 8.57148.66 14.05151.71 12.44PLT(109/
37、L)191.37 38.38238.10 59.04244.25 57.59Triglycerides(mmol/L)a1.21(0.901.58)1.78(1.182.48)2.18(1.633.10)Total cholesterol(mmol/L)4.58 0.924.89 1.035.61 1.26LDL-c(mmol/L)2.53 0.692.83 0.783.24 1.02HDL-c(mmol/L)1.27 0.250.99 0.220.99 0.20FPG(mmol/L)a4.8(4.65.1)5.2(4.96.0)5.4(4.96.2)HbA1c(%)a12.9(11.615.
38、0)5.8(5.46.6)5.9(5.46.6)ALT(U/L)a12(1016)39(2560)61(3890)AST(U/L)a18(1622)29(2335)38(2756)ALP(U/L)67.2 17.574(6592)85(7096)GGT(U/L)a25(2035)44(2774)58(41109)S100A11(ng/mL)5.99(4.229.07)6.59(4.518.79)6.85(4.879.90)BMI=body mass index;BP=blood pressure;WBC=white blood cell;RBC=redblood cell;HGB=Hemogl
39、obin;PLT=platelet;LDL-c=low density lipoprotein cho-lesterol;HDL-c=high density lipoprotein cholesterol;FGP=fasting plasma glu-cose;HbA1c=glycated hemoglobin;ALT=alanine transaminase;AST=aspartateaminotransferase;ALP=alkaline phosphatase;GGT=-glutamyl transpeptidase;Data are shown as mean SD or medi
40、an with interquartile range(IQR)and categoricaldata as n(%).aAnalysis performed on log-transformed data.P 0.05 compared with control.P 0.01 compared with control.F.Teng,J.Jiang,J.Zhang et al.Metabolism Clinical and Experimental 117(2021)1547252Downloaded for Anonymous User(n/a)at Shanghai Jiao Tong
41、University School of Medicine from ClinicalK by Elsevier on April 04,2021.For personal use only.No other uses without permission.Copyright 2021.Elsevier Inc.All rights reserved.GUCUCCGACUGTT;si-NC sense,5-UUCUCCGAACGUGUCACGUTT-3,and anti-sense,5-ACGUGACACGUU CGGAGA ATT-3.2.5.Animal experimentsAll ex
42、periments were approved by the Institute of Animal Care andUse Committee of Xiamen University.Experimental animals(C57BL/6,6-8w,male)were purchased and housed in Xiamen University Labora-tory Animal Center(XMULAC).Animals were fed appropriate food and water ad libitum for twomonths,then S100A11 aden
43、o-associated virus(AAV8)or empty vector(2 1011genome copies per mouse,Vigene Bioscience,Shandong,China)was delivered by tail-veininjection,and experiments were com-menced after 14 days.After four weeks of feeding,the mice weresacrificed,and serum,adipose,and liver tissues were collected andstored in
44、 liquid nitrogen for further experiments.Eight-week-old mice were injected with 2 1011genome copies ofAAV8 encoding a control shRNA or S100A11 shRNA(Vigene Bioscience,Shandong,China).All the mice were fed with HFD after tail-vein injec-tion.Bodyweightandrandombloodglucoseconcentrationsweremon-itored
45、weeklyinallmice.Afterfourteenweeksoffeeding,themiceweresacrificed,and serum,adipose,and liver tissues were collected andstored in liquid nitrogen for further experiments.2.6.Quantitative real-time polymerase chain reactionTotal RNA was isolated from cells or tissue using TRIzol(ThermoFisher Scientif
46、ic,Waltham,MA).The reverse transcription products oftotal RNA from each sample were prepared using the RT reagent kit(Takara,Tokyo,Japan)according to the manufacturers protocol.TheSYBR Real-time PCR Master Mix kit(Yeason,Shanghai,China)wasused for mRNA amplification,and the amplification reactions w
47、ere runon a LightCycler 480 Instrument(Roche,Basel,Switzerland).Data cal-culated by quantitative real-time PCR were analyzed by the 2-Ctmethod.Primers used for the analyses are shown in Table S1.2.7.Western blotCells were lysed with RIPA buffer(Beyotime,Shanghai,China)onice,and supernatantswere harv
48、ested.Cell lysate protein wasquantifiedby the BCA Protein Assay Kit(Pierce)and separated on 10%polyacryl-amide gels,which were then transferred to 0.25 PVDF membranes(Millipore,Billerica,MA).Thenceforth,the membranes were incubatedovernight at 4 C in antibodies for S100A11(1:1000 dilution,Abcam,Camb
49、ridge,MA),RAGE(1:1000,Bioworld,MN,USA),PPAR(1:1000,Abcam,Cambridge,England),G6PC(1:1000,Abcam,Cambridge,En-gland),CD36(1:1000,Proteintech,Chicago,USA),AKT(1:1000,CST,Boston,USA),p-AKT(1:2000,CST,Boston,USA),mTOR(1:1000,CST,Boston,USA),p-mTOR(1:1000,CST),arebp1c(1:500,Santa Cruz,Santa Cruz,USA),or-ac
50、tin(1:10000,Proteintech,Chicago,USA)followed by incubation at room temperature for 1 h in horseradishperoxidase-conjugated secondary antibodies of goat anti-mouse IgG(1:5000 dilution,Abcam,Cambridge,USA)or goat anti-rabbit IgG(1:5000 dilution,RD,Rockford,USA).Membranes were incubatedwith ECL detecti